PROJECT SUMMARY Taste receptor cells (TRCs) are continually replaced from adult stem/progenitor cells, and the fidelity of this process underlies the relative constancy of our sense of taste. However, a host of cancer therapies perturb taste and we posit this is due to perturbation of taste cell renewal. The Wnt/-catenin and Hedgehog pathways are implicated in scores of cancers, and many drugs have been and continue to be developed to target these pathways in tumors; these drugs invariably cause taste dysfunction for patients. Subsets of taste stem cells express the Wnt target gene Lgr5 and the Hedgehog target gene Gli1, and both Wnt and Hedgehog pathways have been shown to regulate taste cell renewal in vivo. Thus, in the long term, understanding the functional relationship of Wnt- and Hedgehog-sensitive stem cells in taste homeostasis, as proposed here, will shed light on how these progenitors are disrupted by chemotherapeutics that cause taste dysfunction, and allow development of strategies to mitigate dysgeusia. In our application, we propose to test explicit hypotheses of the functional relationship of LGR5+ and GLI1+ stem cells in the circumvallate taste papillae of mice. Hypothesis 1: Progenitors expressing high levels of LGR5 are slow cycling, multipotent stem cells that produce rapidly proliferating GLI1+/LGR5low/neg progenitors that give rise directly to TRCs. Hypothesis 2: Upon LGR5+ cell ablation, GLI1+ progenitors expand their potential to reconstitute circumvallate epithelium and give rise to new LGR5+ stem cells. To test these ideas, we combine in vivo molecular genetics, in vitro production of lingual organoids, and single cell transcriptome profiling ? all approaches with which we have become skilled. In Aim 1, we test the competency of LGR5 vs GLI1 progenitors to produce taste cell-replete organoids, and further assess the degree to which lineage production by each progenitor type is dependent upon Wnt signaling. In Aim 2, we explore the capacity of GLI1+ progenitors to regenerate both taste cells and LGR5+ stem cells following genetic ablation of LGR5+ cells. In Aim 3, we combine temporally fine-grained lineage tracing with single cell RNA sequencing to transcriptomically define the cell lineages that continually produce each of the functional taste cell types, i.e., glial-like cells and sweet, bitter, umami, salt and sour TRCs. In sum, our proposed studies will lead to significant advances in our understanding of the cellular and molecular mechanisms that maintain our sense of taste.